Research progress in diseases associated with genetic variants of GATOR1 complex / 中华医学遗传学杂志

The GATOR1 complex is located at the upstream of the mTOR signal pathway and can regulate the function of mTORC1. Genetic variants of the GATOR1 complex are closely associated with epilepsy, developmental delay, cerebral cortical malformation and tumor. This article has reviewed the research progress in diseases associated with genetic variants of the GATOR1 complex, with the aim to provide a reference for the diagnosis and treatment of such patients.

RIP1-dependent linear and nonlinear recruitments of caspase-8 and RIP3 respectively to necrosome specify distinct cell death outcomes

There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions.

The role of the exocyst in renal ciliogenesis, cystogenesis, tubulogenesis, and development

The exocyst is a highly conserved eight-subunit protein complex (EXOC1–8) involved in the targeting and docking of exocytic vesicles translocating from the trans-Golgi network to various sites in renal cells. EXOC5 is a central exocyst component because it connects EXOC6, bound to the vesicles exiting the trans-Golgi network via the small GTPase RAB8, to the rest of the exocyst complex at the plasma membrane. In the kidney, the exocyst complex is involved in primary ciliognesis, cystogenesis, and tubulogenesis. The exocyst, and its regulators, have also been found in urinary extracellular vesicles, and may be centrally involved in urocrine signaling and repair following acute kidney injury. The exocyst is centrally involved in the development of other organs, including the eye, ear, and heart. The exocyst is regulated by many different small GTPases of the RHO, RAL, RAB, and ARF families. The small GTPases, and their guanine nucleotide exchange factors and GTPase-activating proteins, likely give the exocyst specificity of function. The recent development of a floxed Exoc5 mouse line will aid researchers in studying the role of the exocyst in multiple cells and organ types by allowing for tissue-specific knockout, in conjunction with Cre-driver mouse lines.

RASAL1 induces to downregulate the SCD1, leading to suppression of cell proliferation in colon cancer via LXRα/SREBP1c pathway

BACKGROUND: Recent studies have confirmed that RASAL1 has an antitumor effect in many cancers, but its functional role and the molecular mechanism underlying in colon cancer has not been investigated. RESULTS: We collected human colon cancer tissues and adjacent non-tumor tissues, human colon cancer cell lines LoVo, CaCo2, SW1116, SW480 and HCT-116, and normal colonic mucosa cell line NCM460. RT-qPCR was used to detect the RASAL1 level in the clinical tissues and cell lines. In LoVo and HCT-116, RASAL1 was artificially overexpressed. Cell viability and proliferation were measured using CCK-8 assays, and cell cycle was detected via PI staining and flow cytometry analysis. RASAL1 significantly inhibited the cell proliferation via inducing cell cycle arrest, suppressed cell cycle associated protein expression, and decreased the lipid content and inhibited the SCD1 expression. Moreover, SCD1 overexpression induced and downregulation repressed cell proliferation by causing cell cycle arrest. Additionally, luciferase reporter assays were performed to confirm the direct binding between SREBP1c, LXRα; and SCD1 promoter, we also demonstrated that RASAL1 inhibit SCD1 3'-UTR activity. RASAL1 inhibited tumor growth in xenograft nude mice models and shows inhibitory effect of SCD1 expression in vivo. CONCLUSION: Taken together, we concluded that RASAL1 inhibited colon cancer cell proliferation via modulating SCD1 activity through LXRα/SREBP1c pathway.

Structural characterization of coatomer in its cytosolic state

Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry) for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.

Non-catalytic roles for TET1 protein negatively regulating neuronal differentiation through srGAP3 in neuroblastoma cells

The methylcytosine dioxygenases TET proteins (TET1, TET2, and TET3) play important regulatory roles in neural function. In this study, we investigated the role of TET proteins in neuronal differentiation using Neuro2a cells as a model. We observed that knockdown of TET1, TET2 or TET3 promoted neuronal differentiation of Neuro2a cells, and their overexpression inhibited VPA (valproic acid)-induced neuronal differentiation, suggesting all three TET proteins negatively regulate neuronal differentiation of Neuro2a cells. Interestingly, the inducing activity of TET protein is independent of its enzymatic activity. Our previous studies have demonstrated that srGAP3 can negatively regulate neuronal differentiation of Neuro2a cells. Furthermore, we revealed that TET1 could positively regulate srGAP3 expression independent of its catalytic activity, and srGAP3 is required for TET-mediated neuronal differentiation of Neuro2a cells. The results presented here may facilitate better understanding of the role of TET proteins in neuronal differentiation, and provide a possible therapy target for neuroblastoma.

High SIPA-1 expression in proximal tubules of human kidneys under pathological conditions / 华中科技大学学报（医学）（英德文版）

Systemic lupus erythematosus (SLE) and clear cell renal cell carcinoma (CC-RCC) are serious disorders and usually fatal, and always accompanied with pathological changes in the kidney. Signal-induced proliferation-associated protein 1 (SIPA-1) is a Rap1GTPase activating protein (Rap1GAP) expressed in the normal distal and collecting tubules of the murine kidney. Lupus-like autoimmune disease and leukemia have been observed in SIPA-1 deficient mice, suggesting a pathological relevance of SIPA-1 to SLE and carcinoma in human being. The expression pattern of SIPA-1 is as yet undefined and the pathogenesis of these diseases in humans remains elusive. In this study, we used both immunohistochemistry and quantum dot (QD)-based immunofluorescence staining to investigate the expression of SIPA-1 in renal specimens from SLE and CC-RCC patients. MTT assay and Western blotting were employed to evaluate the effects of SIPA-1 overexpression on the proliferation and apoptosis of renal cell lines. Semi-quantitative reverse transcriptase-PCR (RT-PCR) was applied to examine the changes of hypoxia-inducible factor-1α (HIF-1α) mRNA level. Results showed that SIPA-1 was highly expressed in the proximal and collecting tubules of nephrons in SLE patients compared to normal ones, and similar results were obtained in the specimens of CC-RCC patients. Although SIPA-1 overexpression did not affect cellular proliferation and apoptosis of both human 786-O renal cell carcinoma cells and rat NRK-52E renal epithelial cell lines, RT-PCR results showed that HIF-1α mRNA level was down-regulated by SIPA-1 overexpression in 786-O cells. These findings suggest that SIPA-1 may play critical roles in the pathological changes in kidney, and might provide a new biomarker to aid in the diagnosis of SLE and CC-RCC.

The effect of up-regulated expression of Rap1GAP on the invasion ability of HL-60 cells in vitro and in vivo / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of up-regulation of Rap1GAP on the invasion ability of leukemic HL-60 cells in vitro, and to establish leukemia mouse model to verify the effects in vivo.</p><p><b>METHODS</b>Quantitative RT-PCR and Western blot methods were used to detect the expression of Rap1GAP in Venus/HL-60 (vehicle control) and Rap1GAP/HL-60 cells (R1 andR2). Transwell method was used to examine the invasion ability in vitro. Quantitative RT-PCR and gelatin zymograph were used to study the expression of MMP-2 and MMP-9. Four-week-old BALB/c nu/nu mice were pre-treated and inoculated with leukemic cells from different groups, several index including survival time were then monitored.</p><p><b>RESULTS</b>Rap1GAP mRNA level of R1 and R2 increased about 16-17 folds as compared to the control cells. The invasion rate of R1 and R2 are (55 ± 5)% and (59 ± 4)%, which are significantly higher than (14 ± 4)% of the control cells. The mRNA level of MMP-9 was up-regulated about 12.0 folds in R1 and R2 cells compared to the corresponding control cells. The median survival times of R1 and R2 mice are (32.00 ± 1.85) d and (33.37 ± 2.50) d, respectively, which are shorter than (43.62 ± 2.32) d of the control group. Three mice of R1 and R2 groups showed leukemic cells infiltration in meninges tissue, and the genes of Rap1GAP and MMP-9 were amplified by PCR method.</p><p><b>CONCLUSION</b>Up-regulated expression of Rap1GAP increased the invasion ability of HL-60 cells accompanied with enhancement of MMP-9 expression in vitro, and the experiment in mouse model also confirmed that Rap1GAP enhanced the invasion of HL-60 cells in vivo.</p>

Tissue distribution study of salvianolic acid B long-circulating liposomes in mice by UPLC-MS/MS determination

In targeting delivery system research on salvianolic acid B, it's vital but hard to evaluate the tissue distribution for its low concentrations in tissues. So the simple, rapid, selective and sensitive UPLC-MS/MS method was provided hereby to determine the concentration of salvianolic acid B in mice tissues after intravenous administration of salvianolic acid B injections, conventional liposomes and long-circulating liposomes. The UPLC was conducted by a C18 column with a gradient mobile phase consisting of acetonitrile and water containing 0.1% formic acid. The tandem mass spectrometry was operated in negative-electrospray ionization selected-reaction-monitoring mode, and the optimized characteristic precursor to product ion transition m/z 717.3[right wards]519.1 was selected. The biosamples were homogenized and treated with a protein precipitation, which led to an acceptable matrix effect and extraction recovery. The linear calibration curves were plotted in the given concentration ranges. The intra-day and inter-day precisions were less than 13.9% and the accuracies were in the range of 86.3-109.2%. The tissue distribution results determined by UPLC-MS/MS we developed showed that the conventional and long-circulating liposomes we made had succeeded in prolonging the retention time and increasing the level of salvianolic acid B in certain distribution tissues such as liver, kidney and brain

Role and clinical significance of RLIP76 in regulation of multi-drug resistance of small cell lung cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the role of RLIP76 in regulating multi-drug resistance in small cell lung cancer (SCLC), and to analyze the relationship between its expression and prognosis.</p><p><b>METHODS</b>The expressions of RLIP76 protein and gene were detected by Western blotting and real-time PCR (RT-PCR) in both the chemosensitive SCLC H69 cell line and chemoresistant H69AR cell line, respectively. siRNA was transfected into the H69AR cells to inhibit RLIP76 expression, and eGFP-RLIP76 was transfected into the H69 cells to enhance RLIP76 expression. The drug-sensitivity of cells to chemotherapeutic drugs (ADM, DDP, VP-16) were detected by CCK8 assay. The expression of RLIP76 in the SCLC tissues was detected by immunohistochemistry. The relationship of RLIP76 expression with clinicopathological features and prognosis of the patients was analyzed.</p><p><b>RESULTS</b>The expression of RLIP76 in H69AR cells was 13.675 ± 0.983, significantly higher than 1.074 ± 0.107 in the H69 cells (P < 0.01). The drug-sensitivities of H69AR cells to chemotherapeutic drugs were significantly increased when the expression of RLIP76 was down-regulated (P< 0.001). The sensitivities of H69 cells to chemotherapeutic drugs ADM, DDP and VP-16 were significantly decreased after transfection with eGFP-RLIP76 up-regulating the RLIP76 expression (P = 0.003). The positive expression rates were 61.3% and 9.4% in the SCLC tumor tissues and para-cancerous tissues, respectively (P < 0.01). The expression of RLIP76 was significantly correlated with clinical stage, chemosensitivity and overall survival of the SCLC patients (P < 0.05).</p><p><b>CONCLUSIONS</b>Our results suggest that RLIP76 is involved in the regulation of small cell lung cancer multidrug resistance. RLIP76 may serve as a potential target gene to evaluate the chemosensitivity and clinical prognostic for small cell lung cancer.</p>

Role of inhibition of osteogenesis function by Sema4D/Plexin-B1 signaling pathway in skeletal fluorosis in vitro / 华中科技大学学报（医学）（英德文版）

Skeletal fluorosis is a chronically metabolic bone disease with extensive hyperostosis osteosclerosis caused by long time exposure to fluoride. Skeletal fluorosis brings about a series of abnormal changes of the extremity, such as joint pain, joint stiffness, bone deformity, etc. Differentiation and maturation of osteoblasts were regulated by osteoclasts via Sema4D/Plexin-B1 signaling pathway. Furthermore, the differentiation and maturation of osteoclasts are conducted by osteoblasts via RANKL/RANK/OPG pathway. Both of these processes form a feedback circuit which is a key link in skeletal fluorosis. In this study, an osteoblast-osteoclast co-culture model in vitro was developed to illustrate the mechanism of skeletal fluorosis. With the increase of fluoride concentration, the expression level of Sema4D was decreased and TGF-β1 was increased continuously. OPG/RANKL mRNA level, however, increased gradually. On the basis of that, the inhibition of Sema4D/Plexin-B1/RhoA/ROCK signaling pathway caused by fluoride promoted the level of TGF-β1 and activated the proliferation of osteoblasts. In addition, osteroprotegerin (OPG) secreted by osteoblasts was up-regulated by fluoride. The competitive combination of OPG and RANKL was strengthened and the combination of RANKL and RANK was hindered. And then the differentiation and maturation of osteoclasts were inhibited, and bone absorption was weakened, leading to skeletal fluorosis.

Effect of 5-aza-2'-deoxycytidine combined with trichostatin A on RPMI-8226 cell proliferation, apoptosis and DLC-1 gene expression / 中国实验血液学杂志

This study was aimed to investigate the effects of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) and histone deacetylase inhibitor trichostatin A (TSA) on DLC-1 gene transcription regulation and molecular biological behaviours in the human multiple myeloma RPMI-8226 cells. The cells were treated respectively with 5-Aza-CdR and TSA alone, or the both combination; the cell proliferation and apoptosis, DLC-1 expression, the protein expression of Ras homolog family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1) were examined by CCK-8 method, RT-PCR and ELISA, respectively. The results showed that the 5-Aza-CdR and TSA had cell growth inhibitory and apoptosis-inducing effects in dose-dependent manner (P < 0.05). Compared with a single drug (5-Aza-CdR or TSA alone), the effects were significantly enhanced after treatment with the combination of 5-Aza-CdR and TSA (P < 0.05). DLC-1 was weakly expressed in the control group; the treatment with 5-Aza-CdR alone enhanced its re-expression dose-dependently (P < 0.05). Compared with 5-Aza-CdR alone, 5-Aza-CdR plus TSA enhanced DLC-1 re-expression significantly.Compared with the control, 5-Aza-CdR and TSA significantly decreased RhoA and Rac1 protein expression (P < 0.05). It is concluded that 5-Aza-CdR and TSA can effectively reverse DLC-1 expression of RPMI-8226 cells; TSA has a synergistic effect on its re-expression. 5-Aza-CdR and TSA have significant cell growth inhibitory and apoptosis-inducing effects on RPMI-8226 cells. These effects may be related to the inhibition of Rho/Rho kinase signalling pathway.

srGAP3 promotes neurite outgrowth of dorsal root ganglion neurons by inactivating RAC1

OBJECTIVE@#To explore effect of srGAP3 promotes neurite outgrowth of dorsal root ganglion neurons.@*METHODS@#In this study, expression of Slit1 was observed predominantly in the glia, while expression of Robo2 and srGAP3 was detected in sensory neurons of postnatal rat cultured dorsal root ganglion (DRG). Furthermore, upregulation of srGAP3 following sciatic nerve transection was detected by immunohistochemistry and Western blotting.@*RESULTS@#It was observed that inhibition of neurite outgrowth in cultured adult DRG neurons following treatment with anti-srGAP3 or anti-Robo2 was more effectively (1.5-fold higher) than that following treatment with an anti-BDNF positive control antibody. It demonstrated that srGAP3 interacted with Robo2 and Slit1 protein to decrease Rac1-GTP activity in cultured adult rat DRG neurons and the opposite effect on Rac1-GTP activity was detected by co-immunoprecipitation and immunoblotting analyses following treatment with anti-Robo2 or anti-srGAP3. These data demonstrated a role for srGAP3 in neurite outgrowth of DRG sensory neurons.@*CONCLUSIONS@#Our observations suggest that srGAP3 promotes neurite outgrowth and filopodial growth cones by interacting with Robo2 to inactivate Rac1 in mammalian DRG neurons.

XCT790 inhibits rat vascular smooth muscle cells proliferation through down-regulating the expression of estrogen-related receptor alpha / 药学学报

Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an important role in several pathological processes of cardiovascular diseases. In this study, the effects of XCT790, a potent and selective inverse agonist of estrogen-related receptor alpha (ERRalpha), on rat VSMCs proliferation and related signal pathways were investigated. The proliferative activity of VSMCs was determined by CCK-8 assay. The mRNA levels of ERRalpha, PGC-1alpha, OPN and MCAD were assayed by RT-PCR. The protein levels of ERRalpha, ERK2 and p-ERK1/2 were evaluated by Western blotting. ELISA was used to assess the protein expression of VEGF. The results showed that XCT790 (5-20 micromol x L(-1)) inhibited rat VSMCs proliferation, and the expression of ERRalpha and its target genes, as well as p-ERK1/2, were also inhibited. XCT790 inhibited VSMCs proliferation in a dose-dependent manner at the dose range from 5 to 20 micromol x L(-1) and in a time-dependent manner at the dose range from 10 to 20 micromol x L(-1). These findings demonstrate that XCT790 inhibits rat VSMCs proliferation by down-regulating the gene level of ERRalpha and thus inhibiting the ERK signal pathway, suggesting that ERRalpha may be a novel potential target for therapeutic approaches to inhibit VSMCs proliferation, which plays an important role in several cardiovascular diseases.

Receptor interacting protein 1 involved in ultraviolet B induced NIH3T3 cell apoptosis through expression of matrix metalloproteinases and reactive oxygen species production / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Receptor interacting protein 1 (RIP1), which plays a key role in apoptosis, cell survival and programmed cell necrosis, is one of the most important proteins in the RIP family. The purpose of this study was to investigate the roles of RIP1 in the apoptosis, the generation of reactive oxygen species (ROS) and the expression of matrix metalloproteinases (MMPs) induced by ultraviolet B (UVB) in fibroblasts.</p><p><b>METHODS</b>siRNA targeting RIP1 was used to silence RIP1 expression in the NIH3T3 fibroblasts. The mRNA and protein levels of MMP-1 and MMP-3, caspase-3 and -8 activities, and ROS activities were determined by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), immunoblotting, caspase activity assay, immunofluorescence, and flow cytometry.</p><p><b>RESULTS</b>The mRNA and protein expressions of MMP-1 and MMP-3 were significantly increased in RIP1 deficient NIH3T3 cells at 24 hours after UVB treatment. At 24 hours after exposure to UVB, RIP1 deficient NIH3T3 cells presented apoptotic morphology, and the apoptosis rate was significantly increased accompanied by pronounced increase in caspase-8 and -3 activities. ROS production was inhibited by UVB at 12 hours in RIP1 deficient NIH3T3 cells.</p><p><b>CONCLUSION</b>RIP1 is involved in NIH3T3 cell damage induced by UVB via participating in the apoptosis, expression of MMPs and ROS production.</p>

Mechanism underlying the anterograde transport of the influenza A virus transmembrane proteins and genome in host cytoplasm / 生物工程学报

Influenza virus assembly requires the completion of viral protein and vRNP transport to the assembly site at the plasma membrane. Therefore, efficient regulation of intracellular transport of the viral proteins and vRNPs to the surface of the host cell is especially important for virus morphogenesis. Influenza A virus uses the machineries of host cells to transport its own components including ribonucleoproteins (vRNPs) and three transmembrane proteins hemagglutinin (HA), neuraminidase (NA) and matrix 2 protein (M2). It has been shown that newly synthesized vRNPs are associated with active form of Rab11 and accumulate at recycling endosomes adjacent to the microtubule organizing center (MTOC) following nuclear export. Subsequently, they are transported along the microtubule network toward the plasma membranes in cargo vesicles. The viral transmembrane proteins are translated on the rough endoplasmic reticulum and transported to the virus assembly site at the plasma membrane. It has been found that several host factors such as ARHGAP21 and GTPase Cdc42 are involved in regulation of intracellular trafficking of influenza A virus transmembrane proteins including NA. In this review, we will highlight the current knowledge about anterograde transport and its regulation of the influenza A virus transmembrane proteins and genome in the host cytoplasm.

Inhibition of cytohesin-1 by siRNA leads to reduced IGFR signaling in prostate cancer

To explore how cytohesin-1 (CYTH-1) small interfering RNA (siRNA) influences the insulin-like growth factor receptor (IGFR)-associated signal transduction in prostate cancer, we transfected human prostate cancer PC-3 cell lines with liposome-encapsulatedCYTH-1 siRNA in serum-free medium and exposed the cells to 100 nM IGF-1. The mRNA and protein levels of the signal molecules involved in the IGFR signaling pathways were determined by real-time PCR and detected by Western blotting. The relative mRNA levels of CYTH-1, c-Myc, cyclinD1 and IGF-1R (CYTH-1 siRNA group vs scrambled siRNA group) were 0.26 vs 0.97, 0.34 vs 1.06, 0.10 vs 0.95, and 0.27 vs 0.41 (P < 0.05 for all), respectively. The relative protein levels of CYTH-1, pIGF-1R, pIRS1, pAkt1, pErk1, c-Myc, and cyclinD1 (CYTH-1 siRNA group vsscrambled siRNA group) were 0.10 vs 1.00 (30 min), 0.10 vs 0.98 (30 min), 0.04 vs 0.50 (30 min), 0.10 vs 1.00 (30 min), 0.10 vs 1.00 (30 min), 0.13 vs 0.85 (5 h), and 0.08 vs 0.80 (7 h), respectively. The tyrosine kinase activity of IGF-1R was associated with CYTH-1. The proliferative activity of PC-3 cells transfected with CYTH-1 siRNA was significantly lower than that of cells transfected with scrambled siRNA at 48 h (40.5 vs87.6 percent, P < 0.05) and at 72 h (34.5 vs 93.5 percent, P < 0.05). In conclusion, the interference of siRNA with cytohesin-1 leads to reduced IGFR signaling in prostate cancer; therefore, CYTH-1 might serve as a new molecular target for the treatment of prostate cancer.

Expression of deleted in liver cancer 1 and phosphorelated focal adhesion kinase in breast cancer / 南方医科大学学报

<p><b>OBJECTIVE</b>To analyze the expression of deleted in liver cancer 1 (DLC1) and phosphorelated focal adhesion kinase (p-FAK) in breast cancer tissue to further understand the molecular mechanisms of the carcinogenesis and metastasis of breast cancer.</p><p><b>METHODS</b>Immunohistochemistry was employed to determine the protein level of DLC1 and p-FAK in 61 breast cancer, 30 benign breast disease and the adjacent normal breast tissues.</p><p><b>RESULTS</b>The positivity rates of DLC1 differed significantly between breast cancer, benign and normal tissues (34.43%, 80.00% and 76.67%, respectively, P<0.001). The positivity rates of p-FAK in the 3 tissues were 77.05%, 33.33% and 26.67%, also showing significant differences (P<0.001). The aberrant expression of DLC1 showed an inverse correlation to p-FAK (κ=-0.4591). Both DLC1 and p-FAK were closely correlated to the carcinogenesis, clinical stage, PR and lymphatic metastasis of breast cancer (P<0.05), but not to the patients age, pathological subtype, familial history, ER or CerbB-2 (P>0.05).</p><p><b>CONCLUSION</b>The abnormal expression of DLC1 and p-FAK might participate in the carcinogenesis, progression, and metastasis of breast cancer. The role of DLC1 and p-FAK might be related to the regulation of progestone. DLC1 and p-FAK may serve as candidate markers for early diagnosis, prognostic evaluation and target treatment of breast cancer.</p>

Mutational Analysis of the NF1 Gene in Two Families with Neurofibromatosis 1 Accompanied by Pheochromocytoma

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant inherited disorders affecting the nervous system. NF1 is associated with mutations in the NF1 gene, which is located on chromosome sub-band 17q11.2 and contains 57 exons spanning approximately 300 kb of genomic DNA. NF1 is caused by a loss of function mutation of the NF1 gene, a tumor suppressor gene, which encodes for neurofibromin, a GTPase-activating protein (GAP) involved in the negative regulation of Ras activity. The GAP-related domain, which is encoded for by exons 20-27a, is one of the most important functional domains in neurofibromin. The cysteine-serine-rich domain has been recognized as an important functional domain in NF1-related pheochromocytomas. As the result of many genetic analyses of NF1-related pheochromocytomas, pheochromocytoma has generally been recognized as a true component of NF1. We recently experienced two families with NF1 accompanied by pheochromocytoma. The proband of family 1 is a 31-year-old female diagnosed with NF1 and pheochromocytoma. Gene analysis of the proband and her sister showed that the mutation of the NF1 gene (c.7907+1G>A) led to the skipping of exon 53 during NF1 mRNA splicing. The proband of family 2 is a 48-year-old male who was diagnosed with the same condition. Gene analysis demonstrated the mutation of the NF1 gene (c.5206-8C>G) with missplicing of exon 37. These novel germline mutations did not fall into the GAP-related nor the cysteine-serine-rich domains, but into the C-terminal area of the NF1 gene. This suggests that the correlation between the genotype and phenotype of NF1-related pheochromocytoma is somewhat difficult to characterize. Further studies will be necessary to confirm the function of the C-terminal area of the NF1 gene and its contribution to the development of NF1 and pheochromocytoma.